Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using retroviral vectors.

The Moloney murine leukemia retrovirus-derived vector N2 was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells. Approximately 5% of day seven CFU-GM were resistant to 2,000 micrograms/ml G418, using a supernatant infection protocol in the absence of vector-producing cells. A greater proportion of CFU-GM colonies were recovered relative to uninfected controls as the stringency of selection was diminished. Enzyme activity was detected in drug-resistant colonies, confirming that the resistant colonies obtained after infection with N2 represented cells producing neomycin phosphotransferase. Activity in the CFU-GM colonies approached 50% of that of drug-resistant vector-producing cells on a per cell basis. To test the hypothesis that more rapidly cycling bone marrow cells would be more susceptible to vector infection, we treated progenitor cells obtained from cyclic hematopoietic (CH) dogs with the N2 vector. Despite the increased numbers of hematopoietic progenitor cells obtained from CH dogs, the proportion of G418-resistant CFU-GM did not increase over that obtained with N2-infected normal marrow. These results demonstrate that retroviral vectors can be used to transfer and express exogenous genes in canine hematopoietic progenitor cells.

Tritiated thymidine incorporation into canine cyclic hematopoietic bone marrow in vitro.

To investigate changes in the proliferative activity of bone marrow cells in canine cyclic hematopoiesis, nonadherent cells were incubated for 1 h with tritiated thymidine either immediately after the cultures were established or following an 18-h preincubation period. The data suggest that changes in thymidine incorporation show a 12- to 14-day cycle that consists of two distinct phases. During the first six days of the cycle (from peripheral neutropenia to relative neutrophilia), two peaks of incorporation were observed. During the second phase (corresponding to the neutrophilia and oncoming neutropenia), thymidine incorporation was uniformly lower than control values. The change from an apparently cyclical process to a low stable value occurred after the wave of marrow myelopoiesis and close to a time point (days 8-10 of the cycle) at which we have recently suggested significant changes in cell release and/or proliferation take place. The data can be interpreted in the context of a periphery-to-stem-cell feedback loop through an intermediate cell population, probably of myeloid precursors.

Nucleotide and prostaglandin cycling in canine cyclic hematopoiesis.

Bone marrow cells were collected from normal dogs, normal dogs made neutropenic with cyclophosphamide, and 11 dogs affected with cyclic hematopoiesis (CH) on 3 consecutive days of separate 12- to 14-day cycles. The mononuclear marrow cells from both groups of control dogs and from the CH dogs on each of 12-cycle days were cultured for 2.5 hr in serum-free media. The amounts of prostaglandins (PGF2 alpha and PGE) and cyclic GMP (cGMP) measured in the media were found to vary with the cycle in the CH dog. PGF2 alpha was highest as the dogs recovered from the neutropenia and lowest 4 days before the onset of the next neutropenic episode. Cyclic GMP was lowest 4-5 days before the onset of neutropenia, then dramatically increased as the neutropenic period approached. Cyclic GMP was highest when PGF2 alpha was lowest. Normal dogs, made neutropenic with a single dose of cyclophosphamide, had elevations of PGF2 alpha but not PGE or cGMP during the recovery period of active granulopoiesis.

Canine cyclic haematopoiesis: bone marrow adherent cell influence of CFU-C formation.

Canine cyclic haematopoiesis (CH) appears to be a multipotential stem cell defect, possibly due to an intrinsic marrow defect. The in vitro adherent marrow cells of the cyclic haematopoietic (CH) dog were cultured as underlayers beneath normal dog nonadherent marrow cells. The marrow granulocyte-committed colony forming units (CFU-C0 of the normal dog nonadherent cells were cyclically influenced by the CH adherent cell underlayers. The CH adherent cells collected on the ninth or tenth day following the onset of neutropenia did not stimulate CFU-C formation while those collected on the sixth day stimulated as many as 108 colonies. The CH adherent cells collected on other cycle days supported increased CFU-C formation with the exception of cycle day 3 which inconsistently stimulated growth. These data show that the CH marrow in vitro adherent cells alternately stimulate and inhibit in vitro granulopoiesis of normal dog marrows.

The in vitro growth of erythroid colonies from dog bone marrow.

Enriched methyl cellulose media together with either human urinary erythropoietin or serum collected from phlebotomized dogs exposed to hypoxia was used in the study of the erythroid colony forming (CFU-E) capacity of dog marrow. The dog serum erythropoietin was found to be more efficient in stimulating CFU-E than comparable concentrations of human urinary erythropoietin. Numbers of CFU-E were directly related to the culture concentration of the stimulating serum and to the number of cells per plate. Sheep plasma erythropoietin was also found to be effective in stimulating CFU-E growth. The system described is chemically better defined and produced more consistent results than has been reported for the plasma clot method.

Erythroid colony formation in vitro from the marrow of dogs with cyclic hematopoiesis: interrelationship of progenitor cells.

Committed erythroid progenitor cells (Colony Forming Units-Erythroid, CFU-E) have been studied in canine cyclic hematopoiesis (CH) utilizing a semi-solid methyl cellulose culture system. Erythroid colonies were stimulated by the addition of a standard volume of serum from normal dogs that had been phlebotomized and subjected to hypoxia. CFU-E fluctuated over the cycle in dogs with CH from concentrations 4--5 times normal during the periods of peripheral blood neutropenia to less than one tenth of normal during the phases of elevated peripheral blood neutrophil counts. In spite of these marked fluctuations there was no change in the proliferation rate of the CFU-E as estimated by the tritiated thymidine (3H-TdR) suicide technique. Failure to demonstrate a change in the CFU-E proliferation rate may be related to the relative maturity of these cells with the fluctuations in number resulting from a 'feed-in' from more immature cells. The results show that CFU-E fluctuate in the same phase as committed granulocytic progenitor cells (CFU-C). Our current knowledge of the interrelationships of marrow progenitor cells and events in the peripheral blood of dogs with CH is briefly reviewed and some additional questions, raised by recent studies regarding the pathogenesis of this disease, are discussed.

Progenitor cells in canine cyclic hematopoiesis.

Granulocytic (colony-forming units in culture, or CFU-c) and erythrocytic (erythropoietin-responsive cells, or ERC) progenitor cells in canine cyclic hematopoiesis (CH) have been shown to fluctuate over the cycle and in the same phases as one another. The ERC cycle is preceded by 3 or 4 days by a rise in serum erythroid-stimulating activity and is followed by a reticulocytosis. During the cycle CFU-c show a differential sensitivity to two sera, one normal and one containing elevated amounts of colony-stimulating activity. The proliferation rate of CFU-c also fluctuates from well above normal to considerably less than normal over the cycle. These results are discussed in the light of present knowledge of the pathogenesis of canine CH. We suggest that these results support the contention that CH is a disorder of hemopoietic stem cells and that the cycling of humoral factors and peripheral blood cells may follow as a consequence.